Protein kinase Cs (PKCs) are soluble enzymes that are also found associated with the cytoskeleton. One potential mechanism for targeting PKC to specific cytoskeletal structures such as focal contacts and cell- cell junctions is via interactions with other proteins. We have used an overlay assay that detects phosphatidylserine-dependent PKC interactions with other proteins to identify proteins that may mediate PKC binding to cytoskeletal structures. In many cases, the proteins identified by this assay have also been shown to be PKC substrates. The overlay assay was used to screen a lambdagt11 rat kidney library to isolate additional and novel PKC binding proteins/substrates. Two clones, 35A and 35H, have now been characterized. Both are PKC substrates and binding proteins, and phosphorylation decreases PKC binding. Antibodies to 35H recognize an 80 kDa cytoskeletal protein in cultured renal epithelial cells. 35H colocalizes with alpha-PKC at cell-cell junctions. Phorbol esters stimulate a rapid phosphorylation of 35H and its displacement from the cell-cell junctions as well as extensive membrane ruffling. The purpose of this proposal is to use the study of PKC-35H interactions and the effects of 35H phosphorylation as model systems to better define the functions of cytoskeletal-associated PKCs.